Nucleic acids encoding single domain antibodies to chikungunya virus

ABSTRACT

Described herein are nucleic acids encoding single-domain antibodies that might serve as alternatives to conventional monoclonal antibodies for either the detection or treatment of Chikungunya Virus (CHIKV).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application 62/701,301 filed on Jul. 20, 2019 and is a division of U.S. patent application Ser. No. 16/513,881 filed on Jul. 19, 2020, the entirety of each of which is incorporated herein by reference.

FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT

The United States Government has ownership rights in this invention. Licensing inquiries may be directed to Office of Technology Transfer, US Naval Research Laboratory, Code 1004, Washington, D.C. 20375, USA; +1.202.767.7230; techtran@nrl.navy.mil, referencing NC 108,380.

BACKGROUND

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes outbreaks of polyarthritis in humans, and is currently a threat to spread to the United States due to the presence of its mosquito vector, Aedes albopictus.

CHIKV, like other alphaviruses, has a single-stranded positive sense RNA genome of about 11.8 kb encoding two polyproteins: one nonstructural polyprotein that produces four nonstructural proteins involved in genome replication, capping and polyprotein processing and the other structural polyprotein that produces the capsid as well as the E2 and E1 envelope glycoproteins. Virions are produced in the cytoplasm of the infected cells and later are enveloped and budded out of the membrane. Each virion is composed of the genomic RNA with 240 copies of the capsid protein and 240 copies of E1/E2 heterodimers embedded in the membrane. There are 80 trimeric E1/E2 spikes projecting outward from the membrane and the tip of each complex is formed by E2 protein that interacts with the cell receptor. E2 is the target of the most CHIKV-neutralizing antibodies.

Chikungunya virus-like particles (VLPs) are produced by encoding E1/E2 structural proteins with or without capsid proteins from the recombinant plasmids containing the sequences identical to the 3′ region of the genomic RNA. They have been shown to effectively generate immunity in animals and non-human primates. VLPs are a promising vaccine candidate that could provide for the long term solution to CHIKV infection. In addition to developing vaccines, the development of effective small molecule therapeutics and rapid low cost methods for diagnosis are also critical to limiting the severity of the disease and hopefully reducing the long term health effects. Antibody-based therapeutics are being investigated as a way to bridge the gap until an effective immune response occurs. Antibodies can also play a role in the diagnosis of CHIKV infection, as lower cost rapid alternatives to RT-PCR methods.

To date most antibodies investigated for therapeutic or detection applications have been conventional IgGs. As therapeutics, there is the concern with their use, as antibodies are known to exacerbate the illness through an Fc mediated process. As diagnostics, it would be desirable that they be robust enough to be utilized in point-of-care (POC) assays where maintaining a cold chain becomes problematic

It is not believed that a human vaccine or therapeutic is currently available to protect against CHIKV infection. A need exists for techniques to detect CHIKV infection and for an effective therapeutic against the virus.

BRIEF SUMMARY

In one embodiment, an isolated includes a protein sequence selected from the group consisting of SEQ ID Nos. 1-14.

In another embodiment, a method of detecting Chikungunya virus includes analyzing a sample known or suspected to contain Chikungunya virus using an immunoassay comprising an antibody that includes a protein sequence selected from the group consisting of SEQ ID Nos. 1-14, wherein at least a portion of any Chikungunya virus in the sample binds to the antibody, thereby producing a response from the immunoassay indicative of the presence of Chikungunya virus in the sample.

In a further embodiment, a method of inhibiting Chikungunya virus includes contacting Chikungunya virus with an antibody comprising a protein sequence is selected from the group consisting of SEQ ID No. 1 and SEQ ID No. 13.

An additional embodiment includes a nucleic acid encoding a single-domain antibody comprising a protein sequence selected from the group consisting of SEQ ID Nos. 1-14.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing antibodies and recombinant derivatives. Other than sdAb, all those depicted exhibit a multi-domain nature. The heavy variable domains are shown in black, light domains in grey and the constant domains in white.

FIG. 2 shows protein sequences of potential CHIKV-binding single domain antibodies.

FIG. 3 displays selected CHIKV-binding single domain antibody protein sequences.

FIGS. 4A-4C provide plasmon resonance binding affinity data.

FIGS. 5A and 5B present results of a MagPlex Sandwich assay for CHIKV VLPs using sdAb.

FIGS. 6A-6C show results of an enzyme-linked immunosorbent assay (ELISA) assay for CHIKV VLPs using sdAbs and a monoclonal CHK48.

FIGS. 7A and 7B show CC3 and CA6 dose dependent inhibition of Vero cell infection by CHIKV.

DETAILED DESCRIPTION

Definitions

Before describing the present invention in detail, it is to be understood that the terminology used in the specification is for the purpose of describing particular embodiments, and is not necessarily intended to be limiting. Although many methods, structures and materials similar, modified, or equivalent to those described herein can be used in the practice of the present invention without undue experimentation, the preferred methods, structures and materials are described herein. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

As used herein, the singular forms “a”, “an,” and “the” do not preclude plural referents, unless the content clearly dictates otherwise.

As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

As used herein, the term “about” when used in conjunction with a stated numerical value or range denotes somewhat more or somewhat less than the stated value or range, to within a range of ±10% of that stated.

Overview

For detection applications many immunoassays rely on monoclonal or polyclonal antibodies (IgG) derived from mice, rabbits, goats, or sheep as recognition elements. Functional IgG are comprised of four polypeptide chains, two identical heavy (H) chains and two identical light (L) chains, linked by disulfide bonds. Each antibody has two antigen binding domains formed by the interaction of adjacent variable (V) domains from the H and L chains. The antigen binding surface is composed of six complementarity-determining regions (CDRs), three residing in each of the VII and VL protein domains. The interaction of these six CDR loops of varying sizes and sequences allows the formation of diversified antigen binding surfaces with the topologies to recognize a wide range of antigenic targets. Although sensitive and specific, conventional antibodies can be time-consuming and expensive to develop and have limited stability. FIG. 1 shows a schematic representation of IgG as well as the cloned binding derivative. Cloned derivatives of conventional IgG, comprising just the VII and VL domains to form a minimal antigen binding construct have been used as recognition elements for biosensor applications. These single chain antibodies (scFv) can be expressed in bacteria and modified by protein engineering to tailor the functionality and properties of the antibody fragments. ScFv, however, are often less stable than the parental full-length antibodies and just like full-length antibodies, they aggregate irreversibly at elevated temperatures due to their two-domain structure. Ideally, development of a single-domain structure capable of antigen binding would avoid aggregation upon heating and would facilitate the application of biosensors at elevated environmental temperatures or for continuous use over long periods of time.

Certain animals, such as camelids (i.e. camels, llamas) and sharks, possess a class of immunoglobulins consisting of heavy-chain homodimers where antigen binding is mediated through a single V domain. These V domains, when cloned as single domain antibodies (sdAb), comprise the smallest known antigen binding fragments (12-15 KDa). Despite their small size, sdAb display a high level of specificity and affinity for their antigens and have been shown to have nanomolar affinities (KD) for haptens and proteins. SdAb can re-fold to bind antigen after chemical or heat denaturation enabling them to retain the ability to bind antigen after exposure to elevated temperatures. Several studies have found sdAbs to be inherently thermostable, demonstrating antigen binding at elevated temperatures, which suggests they will be well suited for long-term field applications where refrigeration is often not possible. Recognition elements based on sdAb should offer the specificity of conventional antibodies with the potential for use and storage at elevated temperatures and the regeneration of sensor surfaces.

SdAb also offer several attractive features for therapeutic applications. Again their stability can make them stable at room temperature whereas most antibody therapeutics require refrigeration. Their small size means that the same weight of protein will have 5 times the specific activity and will diffuse much faster throughout the tissue. Its lack of an Fc domain may also be highly beneficial for the case of CHIKV therapeutics where the Fc mediated effects of the immune response appear to be responsible for much long term negative impact of CHIKV infection, namely the polyarthritis.

Examples

Llama-derived sdAb against Chikungunya Virus (E1 and virus like particles (VLPs)) were selected from a library originating from CHIKV VLP immunized animals. The binding ability of these isolated sdAb was characterized by a combination of surface plasmon resonance, enzyme linked immunosorbent assay (ELISA) and Luminex-based MagPlex assays. The sdAb were specific for either the E1 protein or bound both the E1 protein and VLPs. When two of these (CA6 and CC3) were tested in an assay to measure their ability to prevent the viral infection of Vero cells, both sdAb were effective in inhibiting cell infection. Single domain antibodies are often more stable than conventional antibodies and could be formulated to be delivered to resource limited areas that lack the refrigeration required for conventional immunoreagents.

Fourteen sdAb were selected from a library constructed from immunized llamas (FIG. 2 ). They have the following sequences:

CC3 (SEQ ID No: 1): EVQLQASGGGSVQAGGSLRLSCVTSQNLFEYYTMGWYRQVPGSQRERVA LINNGGSTVAGSVEGRFTISRDHAKNSVYLQMNYLKPEDSAVYYCRAFG PADYWGQGTQVTVSS CG6 (SEQ ID No: 2): EVQLQASGGGLVQPGGSLRLSCVASQNLFEYYTMGWYRQVPGSQRERVA LINNGGSNVAGSVEGRFTISRDNTKNSIYLQMNNLKPEDSAVYYCRAFG PADYWGQGTQVTVSS CH5 (SEQ ID No: 3): EVQLQASGGGSVQAGGSLRLSCVASQNLFEYYTMGWYRQVPGSQRERVA LINNGDSNVAGSVEGRFTISRDNAKNSIYLQMNNLKPEDSAVYYCRAFG PADYWGQGTQVTVSS CG1 (SEQ ID No: 4): EVQLQASGGGSVQAGGSLRLSCVASQNLFEYYTMGWYRQVPGSQRERVA LINNGGSNVAGSVEGRFTISRDNAKNSIYLQMNNLKPEDSAVYYCRAFG PADYWGQGTQVTVSS CC2 (SEQ ID No: 5): EVQLQASGGGSVQAGGSLRLSCVASQNLFEYYTMGWYRQVPGSQRERVA LINNGGSNVAGPVEGRFTISRDNAKNSIYLQMNNLKPEDSAVYYCRAFG PADYWGQGTQVTVST CH2 (SEQ ID No: 6): EVQLQASGGGSVQAGGTLRLSCVSSQNLFEYYTMSWYRQVPGSQRERVA LINNGGSDVAGSVEGRFTISRDNAKNSIYLQMNNLKPEDSAVYYCRAFG PADYWGQGTQVTVSS CF2 (SEQ ID No: 7): EVQLQASGGGSVQAGGSLRLSCVSSQNLLEYYTMGWYRQVPGSQRERVA LINNGGSNVAGSVEGRFTISRDNAKNSIYLQMNNLKPEDSAVYYCRAFG PADYWGQGTQVTVSS CD11 (SEQ ID No: 8): DVQLQASGGGLVQAGGTLRLSCAHSGRTSSTQFWGWFRQAPGKEREFVA GMSRSGLSTFYADSVKGRFAISRDSGKNTVYLQMNSLKPEDTAVYFCAS SPFIGEHYYSSTKYHYWGQGTQVTVSS CC12 (SEQ ID No: 9): EVQLQASGGGLVQAGGTLRLSCAHSGRTSSTQFWGWFRQAPGKEREFVA GMSRSGLSTFYADSVKGRFAISRDNGKNTVYLQMNSLKPEDTAVYFCAS SPFIGEHYYSSTKYHYWGQGTQVTVSS CB11 (SEQ ID No: 10): DVQLQASGGGLVQAGGTLRLSCAHSGRTSSTQFWGWFRQAPGKEREFVA GMSRSGLSTFYADSVKGRFAISRDNGKNTVYLQMNSLKPEDTAVYFCAS SPFIGEHYYSSTKYHYWGQGTQVTVSS CE7 (SEQ ID No: 11): EVQLQASGGGLVQAGGTLRLSCAHSGRTSSTQFWGWFRRAPGKEREFVA GMSRSGLSTFYADSVKGRFAISRDNGKNTVYLQMNSLKPEDTAVYFCAS SPFIGEHYYSSRKYHYWGQGTQVTVSS CH6 (SEQ ID No: 12) EVQLQASGGGLVQAGGSLRLSCAASQNIFSINVMGWYRQAPGEQRELVA AITSGGSTNVADSVKGRVTISRDNAKNTVYLQMNSLKPEDTAVYYCAAE ETYYSGSYYGDMEYWGQGTQATVSS CA6 (SEQ ID No: 13): EVQLQASGGGLVRPGGSLRLSCAASGSFFTIDTMAWYRQAPGRRRELVA RQSSGRSPDYDDSVVGRFTISRDIAKSSVYLQMDSLQPEDTALYYCYQS IRPWPGSSYEAHWGQGIQVIVSS CC5 (SEQ ID No: 14): EVQLQASGGGLVQPGGSLRLSCAASGSFFTIDTMAWYRQAPGKQRELVA RQSSGRSPDYDDSVVGRFTISRDIAKSSVCLQMDSLQPEDTALYYCYQS IRPWPGSSYEAHWGQGIQVIVSS

FIG. 2 shows an alignment of these sequences and a consensus sequence (SEQ ID NO: 15).

Five clones were selected from those four sequence families and deemed to be representative with regard to specificity and affinity (FIG. 3 , including a consensus sequence with SEQ ID NO: 16). The binding affinity (K_(D)) was determined by surface plasmon resonance. The two binders shown have good affinity for CHIKV (low nM KD), with CC3 binding to the E1 protein immobilized in land 1 (L1) and the CHIKV VLP immobilized in lane 2 (L2), while CA6 was only observed to bind to the E1 protein in this assay.

The binding affinity (KD) for all the binders was determined by surface plasmon resonance. The two binders shown have good affinity for CHIKV (low nM KD), with CC3 binding to the E1 protein immobilized in land 1 (L1) and the CHIKV VLP immobilized in lane 2 (L2), while CA6 was only observed to bind to the E1 protein in this assay. These data are provided in FIGS. 4A-4C.

MagPlex sandwich immunoassays were used for CHIKV VLPs using all five of the prepared sdAb. This showed that using both CC3 and CA6 as the biotinylated tracer that CC3 and the other member of that family CH5 acted as the strongest capture sdAb for the CHIKV VLPs as seen in FIGS. 5A and 5B.

ELISAs were also evaluated using CA6 and CC6 along with an anti CHIKV monoclonal antibody (CHK48) to demonstrate they would work in this format. FIGS. 6A-6C show that, as in the MagPlex assay, CC3 (also called C3C) performed better than CA6.

The ability of the sdAb to inhibit the CHIKV was examined. Twelve two-fold serial dilutions of each sdAb (CC3, 39 μg/mL and CA6, 10 μg/mL) starting at the respective concentrations were prepared. Each dilution was incubated with ˜300 plaque forming units (PFU) of CHIKV in duplicate for one-hour inoculation. The compound-virus mix was then added to Vero cells seeded in 24-well culture plates for one-hour incubation, followed by adding 0.8% methylcellulose to each well and incubating for three days at 37° C. in a humidified 5% CO2 atmosphere.

On day three post incubation, the Vero cells were fixed and infected foci were counted by using crystal violet staining. 50 percent plaque reduction neutralization titer (PRNT50) of each compound was calculated using XLfit dose response model. The results, seen in FIGS. 7A and 7B, show that CC3 (C3C) is highly effective at inhibiting the infection of Vero cells by CHIKV. CA6 was also effective, but required higher doses to do so.

Further Embodiments

Also contemplated herein are additional antibodies that incorporate the consensus sequences of FIGS. 2 and 3 .

In addition to the assays used in the above examples, other assays incorporating anti-CHIKV sdAbs are contemplated. Such assays might be used, for example, to detect whether a patient might be infected with CHIKV.

It is further contemplated that such antibodies might be active ingredients in treatments for CHIKV infection.

Advantages

These new sequences represent rugged detection reagents that have potential uses as therapeutics. Whereas conventional antibodies will require cold storage and cannot be easily tailored to work optimally with various detection platforms, sdAb are rugged binding molecules can be engineered to be even more thermally stable if need be, but they naturally have robust stability being able to refold if denatured, and they can also be expressed with a variety of fusion domains to enhance their utility. In addition, one of the lasting problems associated with CHIKV infection is the instigation of long lasting arthritic conditions, while it is unclear if these sdAb could help prevent that sequelae, they are an attractive alternative to be investigated.

Concluding Remarks

All documents mentioned herein are hereby incorporated by reference for the purpose of disclosing and describing the particular materials and methodologies for which the document was cited.

Although the present invention has been described in connection with preferred embodiments thereof, it will be appreciated by those skilled in the art that additions, deletions, modifications, and substitutions not specifically described may be made without departing from the spirit and scope of the invention. Terminology used herein should not be construed as being “means-plus-function” language unless the term “means” is expressly used in association therewith.

REFERENCES

-   Pal P, Dowd K A, Brien J D, Edeling M A, Gorlatov S, et al. (2013)     “Development of a Highly Protective Combination Monoclonal Antibody     Therapy against Chikungunya Virus.” PLoS Pathog 9(4): e1003312.     doi:10.1371/journal.ppat.1003312 -   Warter L, Lee C Y, Thiagarajan R, Grandadam M, Lebecque S, et al.     “Chikungunya Virus Envelope-Specific Human Monoclonal Antibodies     with Broad Neutralization Potency.” J Immunol 186 (5), 2011. -   Rebecca Broeckel R, Fox J M, Haese N, Kreklywich C N,     Sukulpovi-Petty S, et al. “Therapeutic administration of a     recombinant human monoclonal antibody reduces the severity of     chikungunya virus disease in rhesus macaques” PLoS Negl Trop Dis     11(6): e0005637, 2017. -   US 2016/0145323A1 “Antibodies against chikungunya virus and uses     thereof,” Doranz B, Mattia K, Kahle K, Simmons G. May 26, 2016 

What is claimed is:
 1. A nucleic acid encoding a single-domain antibody comprising a protein sequence selected from the group consisting of SEQ ID Nos: 1-14.
 2. The nucleic acid of claim 1, wherein the protein sequence is selected from the group consisting of SEQ ID No: 1 and SEQ ID No:
 13. 3. A nucleic acid encoding a single-domain antibody comprising a protein sequence having SEQ ID No:
 1. 